This year in the U.S. ~40,000 new cases of renal cell carcinoma (RCC) will be diagnosed and >13,000 deaths are expected from RCC. Metastatic RCC carries a median survival of 8 months and ~30% of RCC patients are diagnosed with advanced metastatic disease. Localized RCC is treated surgically through resection; however, there is no efficacious therapy currently available for metastatic disease. Adaptive immunity to RCC plays a critical role in tumor surveillance, with both CD8+ and CD4+ T cells contributing to tumor regression. One of the major sites of RCC metastasis is the lung, an environment that is known to be immunosuppressive even in the absence of tumor outgrowth. Therefore, if novel immunotherapies against metastatic RCC are to move forward into clinical trials, the effect of the lung environment on the generation and maintenance of protective T cell responses, compared to the primary tumor site, must be understood. We previously described the development of a recombinant adenoviral vector encoding the human TNF-related apoptosis-inducing ligand (TRAIL; Ad5-TRAIL) cDNA. More recently, our studies have focused on using a similar recombinant adenovirus encoding full-length murine TRAIL (Ad5-mTRAIL) in immunocompetent mice bearing experimental renal cell carcinoma (Renca) tumors. Antigens derived from Ad5-mTRAIL-killed Renca cells could be cross- presented to CD8+ T cells in vitro, and Ad5-mTRAIL used alone minimally prolonged survival and induced a low level of CTL activity in vivo. To enhance DC efficiency we combined Ad5-mTRAIL with CpG oligonucleotides (CpG), which significantly augmented in vivo antigen-specific T cell proliferation and CTL activity, as well as prolonged survival of Renca tumor-bearing mice. Interestingly, CD4+ or CD25+ cell depletion prior to therapy further enhanced survival and in vivo CTL activity, and CD4+ cell-depleted, tumor-free mice also rejected a subsequent challenge of Renca cells, but not MHC-matched prostate tumor cells, showing the existence of tumor-specific immunologic memory. While the effects of CD4+ cell depletion were striking, survival did not reach 100%, suggesting the presence of additional immunosuppressive mechanisms. In this renewal application, we propose the following specific aims to investigate the hypothesis that localized Ad5-TRAIL administration, when used in conjunction with therapies designed to overcome tumor- derived immunosuppression, will lead to protective antitumor immunity in the lungs of mice with experimental metastatic RCC: Aim 1) Ascertain the extent to which intratumoral Ad5-mTRAIL/CpG administration at the primary RCC tumor site impacts the magnitude of CD8+ effector T cell responses against experimental lung metastases; Aim 2) Examine the effect of CD4+ T cell subsets, specifically CD4+CD25+ Treg and Th17, on the quality and quantity of the CD8+ T cell-mediated antitumor response against RCC lung metastases; and Aim 3) Examine the impact of tumor-infiltrating myeloid cell populations on the lung antitumor immune response during Ad5-mTRAIL/CpG immunotherapy - focusing on the stimulatory versus suppressive capacities of these cells. The current proposal reflects our commitment to develop novel immunotherapies for RCC that can eliminate both primary tumors and distal metastases, thereby prolonging the survival of patients with advanced RCC.